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1.
Proc Natl Acad Sci U S A ; 116(29): 14614-14619, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31262815

RESUMO

Aberrant MYC oncogene activation is one of the most prevalent characteristics of cancer. By overlapping datasets of Drosophila genes that are insulin-responsive and also regulate nucleolus size, we enriched for Myc target genes required for cellular biosynthesis. Among these, we identified the aminoacyl tRNA synthetases (aaRSs) as essential mediators of Myc growth control in Drosophila and found that their pharmacologic inhibition is sufficient to kill MYC-overexpressing human cells, indicating that aaRS inhibitors might be used to selectively target MYC-driven cancers. We suggest a general principle in which oncogenic increases in cellular biosynthesis sensitize cells to disruption of protein homeostasis.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Fatores de Transcrição/metabolismo , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Células Epiteliais , Feminino , Humanos , Insulina/metabolismo , Masculino , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética
2.
Proc Natl Acad Sci U S A ; 115(18): 4719-4724, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29666231

RESUMO

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in Drosophila melanogaster We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE.


Assuntos
Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Proteínas de Drosophila , Regulação da Expressão Gênica/genética , Fatores de Transcrição , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética
3.
Proc Natl Acad Sci U S A ; 114(35): 9409-9414, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28808002

RESUMO

While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.


Assuntos
Sistemas CRISPR-Cas , Drosophila melanogaster/genética , Fatores de Transcrição/genética , Ativação Transcricional/genética , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genoma , Genótipo , Larva , RNA/genética , RNA/metabolismo
4.
Nat Methods ; 8(5): 405-7, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21460824

RESUMO

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.


Assuntos
Drosophila melanogaster/genética , Genoma de Inseto , Interferência de RNA , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Técnicas Genéticas , Vetores Genéticos , MicroRNAs/genética , Oogênese/genética , RNA Interferente Pequeno/genética
5.
Nat Commun ; 14(1): 2162, 2023 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-37061542

RESUMO

Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.


Assuntos
Proteínas de Drosophila , Mapas de Interação de Proteínas , Animais , Mapas de Interação de Proteínas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mapeamento de Interação de Proteínas/métodos , Técnicas do Sistema de Duplo-Híbrido
6.
Curr Protoc Mol Biol ; 130(1): e112, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31869524

RESUMO

The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS.


Assuntos
Sistemas CRISPR-Cas , Drosophila/citologia , Drosophila/genética , Técnicas de Introdução de Genes/métodos , Genes de Insetos , Proteínas de Fluorescência Verde/genética , Fases de Leitura Aberta , Animais , Células Cultivadas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes/métodos , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genética , Transfecção
7.
Genetics ; 214(4): 755-767, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32071193

RESUMO

The Transgenic RNAi Project (TRiP), a Drosophila melanogaster functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most Drosophila genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering.


Assuntos
Animais Geneticamente Modificados/genética , Bases de Dados Genéticas , Drosophila melanogaster/genética , Animais , Sistemas CRISPR-Cas , Mutação com Ganho de Função , Engenharia Genética/métodos , Mutação com Perda de Função
8.
Dev Biol ; 315(2): 521-34, 2008 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-18241855

RESUMO

Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins, CRX, OTX1, and OTX2, fulfill these functions. In Drosophila, the single founding member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. We have used global gene expression analysis in late pupal heads to better characterize the post-embryonic functions of Otd in Drosophila. We have identified 61 genes that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one-third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well-documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes, notwithstanding the significant divergence of the underlying genetic pathways. Our findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Transativadores/genética , Transativadores/metabolismo , Vertebrados/genética , Vertebrados/metabolismo , Animais , Animais Geneticamente Modificados , Ritmo Circadiano/genética , Drosophila/crescimento & desenvolvimento , Evolução Molecular , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Óperon Lac , Transdução de Sinal Luminoso/genética , Masculino , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Células Fotorreceptoras de Invertebrados/crescimento & desenvolvimento , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Especificidade da Espécie , Vertebrados/crescimento & desenvolvimento
9.
Elife ; 82019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31674908

RESUMO

We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas de Introdução de Genes/métodos , Recombinação Homóloga , Mutagênese Insercional/métodos , Animais , DNA/genética , DNA de Cadeia Simples/genética , Drosophila
10.
Genetics ; 201(3): 843-52, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26320097

RESUMO

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).


Assuntos
Drosophila/genética , Interferência de RNA , Acesso à Informação , Animais , Animais Geneticamente Modificados , Pesquisa Biomédica , Boston , Genes de Insetos , Vetores Genéticos , Faculdades de Medicina
11.
Dev Cell ; 28(4): 459-73, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24576427

RESUMO

Stem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.


Assuntos
Diferenciação Celular , Divisão Celular/fisiologia , Linhagem da Célula/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Germinativas/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Feminino , Células Germinativas/metabolismo , Ovário/citologia , Ovário/metabolismo , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo
12.
Dev Biol ; 286(1): 158-68, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16125693

RESUMO

The development of the Drosophila visual system utilizes two members of the highly conserved Six-Homeobox family of transcription factor, Sine oculis and Optix. Although in vitro studies have detected differences in DNA-binding and interactions with some co-factors, questions remain as to what extent the activity for these two transcriptional regulators is redundant or specific in vivo. In this work, we show that the SoD mutation within the Six domain does not abolish DNA-protein interactions, but alters co-factor binding specificity to resemble that of Optix. A mutation in the same region of Optix alters its activity in vivo. We propose that the dominant mutant phenotype is primarily due to an alteration in binding properties of the Sine oculis protein and that distinct partner interactions is one important mechanism in determining significant functional differences between these highly conserved factors during eye development.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/crescimento & desenvolvimento , Drosophila/fisiologia , Proteínas do Olho/fisiologia , Olho/crescimento & desenvolvimento , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência Conservada , DNA/genética , DNA/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Dominantes , Genes Homeobox , Genes de Insetos , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Ligação Proteica , Fatores de Transcrição/genética
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